Guanine nucleotide-binding proteins are critical elements in many signal transducing and metabolic pathways in animal cells. One of these proteins, ADP-ribosylation factor, was identified based on its ability to enhance, in the presence of GTP, the ADP-ribosyltransferase activity of cholera toxin, a reaction believed to be important in the pathogenesis of cholera. These studies were consistent with the conclusion that ARF serves as an allosteric activator of the toxin. To study in more detail the effects of ADP-ribosylation factor, a recombinant protein was synthesized in E. coli. The recombinant ARF exhibited high affinity guanine nucleotide-binding and stimulated the ADP-ribosyltransferase activities of cholera toxin and E. coli heat-labile enterotoxins LT-I, LT-IIA and 11 LT-IIB. The E. coli heat-labile enterotoxins were previously shown to catalyze enzymatic reactions similar to those of cholera toxin and to have similar effects on target tissues. Cholera toxin and the E. coli toxins exhibit considerable conservation in structure, particularly in the catalytic subunit. These studies are consistent with the conclusion that both the catalytic activity and the allosteric sites are conserved in the toxins.